Cell-Scaffold Constructs for Bone Regeneration Therapy: Comparison
View Latest Version
Please note this is a comparison between Version 2 by Lindsay Dong and Version 1 by Venkata Suresh Venkataiah.

Bone tissue engineering (BTE) is a process of combining live osteoblast progenitors with a biocompatible scaffold to produce a biological substitute that can integrate into host bone tissue and recover its function. Mesenchymal stem cells (MSCs) are the most researched post-natal stem cells because they have self-renewal properties and a multi-differentiation capacity that can give rise to various cell lineages, including osteoblasts. BTE technology utilizes a combination of MSCs and biodegradable scaffold material, which provides a suitable environment for functional bone recovery and has been developed as a therapeutic approach to bone regeneration.

  • bone tissue engineering
  • MSCs
  • osteoblasts
  • scaffolds

1. Introduction

Continuous research is ongoing in bone tissue regeneration technologies related to orthopedics and dentistry. Vast challenges remain, however, in the application of these modalities to reconstituting damaged skeletal structures. Bone grafting has been widely utilized as a regenerative therapy for critical size bone defects (CSDs), and various bone grafting and prosthetic bone materials have been developed in this regard. There is no one standard definition of CSDs. In general, a “critically-sized” defect is regarded as one that would not heal spontaneously within a patient’s lifetime and would require surgical stabilization and further surgical intervention [1,2][1][2]. Currently, bone grafting materials are classified as autogenous, allogeneic, or heterogeneous and artificial bone substitutes such as hydroxyapatite (HA), β-TCP (beta-tricalcium phosphate), bioactive glass, and calcium sulfate. Autologous bone has no particular disadvantages other than restrictions on the collection amount and collection site and is recognized as a good prosthetic material with new bone formation capacity. It is thus considered the current gold standard for the regeneration of bone defects but has been most widely used in clinics to treat only small-sized bone defects [3,4,5][3][4][5].
To overcome the limitations of current bone graft therapies, such as autologous bone graft and artificial bone substitutes, many researchers have attempted to develop BTE to regenerate and restore lost bone tissue using MSCs, growth factors, and scaffolds [6,7,8,9,10][6][7][8][9][10]. MSCs are referred to as multipotential progenitor cell populations that can differentiate into osteoblast progenitors in vitro under specific conditions, and these cells are most commonly used for bone regeneration [11]. In addition, MSCs are immune tolerant and are used for immunosuppressive therapy via allogenic applications to accelerate bone healing [12]. The use of a scaffold can provide the space needed to deliver and confine MSCs to the bone target site, provide an environment suitable for the migration, proliferation, and differentiation of the stem cells, enable diffusion of nutrients and eventually create early osteoid tissue at the site of the defect which is subsequently mineralized to form new bone. This combination of MSCs and scaffolds has been developed as a BTE therapy. Clinical trials for recovering bone defects have already commenced and reported the accelerated bone healing ability of these approaches. The current bone regenerating ability of the BTE approach is therefore successful but cannot as of yet recover the functional loss caused by large bone defects, such as those resulting from inflammatory diseases.
Osteoblasts are bone-forming cells derived from multipotent mesenchymal stem cells. During skeletal development, multipotent mesenchymal stem cells differentiate into osteoblast progenitor cells and undergo a commitment to form immature osteoblasts that are capable of proliferating before becoming mature osteoblasts. Although mature osteoblasts can synthesize and deposit bone extracellular matrix components, their ability to proliferate is significantly reduced [13]. Thus, recapitulating immature osteoblast differentiation has been suggested as a potential approach to bone regeneration therapy [14]. Currently, osteoblast progenitor cells can be isolated from adult human tissue and are good alternatives to MSCs for bone regeneration. BTE using immature osteoblast and bioscaffolds is, therefore an alternative tissue-engineered construct for recovering large bone defects.

2. MSCs Derived from Embryonic Stem Cells and Induced Pluripotent Stem Cells for Bone Tissue Regeneration

Functional bone tissue engineering generally involves the use of osteoprogenitors derived from MSCs and seeded onto a scaffold to predictably restore the lost architecture and function of bone tissue. MSCs have been isolated from adult tissues such as adipose tissue, bone marrow, and dental tissues, are widely used in regenerative medicine, including BTE, and, thus, have both research and clinical applications. However, MSCs cannot be isolated from patients with systemic disorders such as cardiovascular disease, diabetes, inflammatory bone disease, or advanced aging-related issues. Embryonic stem cell (ESCs)- or Induced pluripotent stem cell (iPSCs)-derived MSCs may be potential cell sources for the clinical trial of BTE [17][15]. A better understanding of cell fate decisions and differentiation processes during osteoblast development may help to generate functional progenitor cells for tissue restoration. Over the years, technologies involving the osteoblast differentiation of ESCs and iPSCs have been significantly improved, and several studies have demonstrated the successful production of MSCs derived from ESCs/iPSCs for use in BTE therapies [18,19,20][16][17][18] (Figure 1).
Cells 10 02687 g001 550
Figure 1. Schematic diagram of different approaches to obtain Mesenchymal stem cells. MSCs can be derived from either iPSCs, ESCs, or adult mesenchymal tissue. MSCs can be obtained by ESCs and iPSCs using small molecules such as mitogen-activated protein kinase (MEK) inhibitor, (MEK) inhibitor, PD0325901, glycogen synthase kinase 3 (GSK3) inhibitor, and CHIR99021 (CHIR). MSCs are also be derived from various connective tissues such as bone marrow, adipose tissue, and dental tissues by collagenase digestion or aspirates from bone marrow and adipose tissue directly used for BTE therapeutics. KLf4: Kruppel Like Factor 4, Oct4: Octamer-binding transcription factor 4, C-myc: Cellular-Myelocytomatosis, Sox-2: sex-determining region Y-box 2.

3. MSCs for Bone Regeneration

To develop MSCs that have clinical utility for BTE, a standard protocol for the characterization, osteoblast differentiation, and transplantation of these cells in combination with a biodegradable scaffold is required. Various types of MSCs are currently available with osteoblastic lineage differentiation potential; however, their origin and development are not clearly understood. There have been few reports on MSCs being successfully derived from neural crest cells during the development of vertebrates, which is seen as transient embryonic tissue [29][19]. Most studies on MSCs to date have reported their derivation from perivascular cells, the pericytes. These cells reside in specific niches, which are commonly found in bone marrow, adipose tissue, and various fetal and other adult tissues [30,31][20][21]. This has been the primary cell source of the MSCs used in BTE to date.

3.1. Characterization of MSCs

Surface markers are currently being used to identify MSCs for quality control assurance prior to cell preparation, based on ‘good manufacturing practice,’ which is required for investor-mediated clinical developments. Hence, the characterization of MSCs based on surface marker analysis is an essential criterion for the clinical application of BTE methodologies. According to the International Society of Cell Therapy (ISCT) criteria, MSCs express a cluster of differentiation (CD) surface markers such as CD90, CD105, and CD73, but do not express CD11b, CD14, CD19, CD34, CD45, or human leukocyte antigen (HLA)-DR [32,33,34][22][23][24].

3.2. Clinical Translation of MSC-Based Bone Regeneration

The basic concept behind a scaffold is to mimic the structure and function of the extracellular matrix (ECM) in tissues. The ECM provides both structural and mechanical stability and regulates some of the core cellular functions [42,43,44][25][26][27]. The basic role of scaffolds in BTE is to mimic the ECM of the native bone tissue and provide a functional three-dimensional space for the adhesion, migration, proliferation, and differentiation of osteoblast progenitors in which bone growth can occur [38,45,46,47][28][29][30][31]. An ideal scaffold for BTE should substitute for both the structure and function of the ECM and thus be capable of regenerating the lost bone tissue when seeded in conjunction with osteoblast progenitors. BTE innovations have led to the development of new biomaterials that resemble the 3D bone structure, in terms of mechanical properties as well as osteoconductive, osteoinductive, and osteogenic features [48,49][32][33]. Traditional bone repair approaches mainly focus on the use of bone grafts from autologous, allogeneic, and xenogeneic sources; however, complications such as donor-site morbidity and host immune rejection limit the application of these tissues [50][34]. The promise of BTE has principally involved overcoming these problems. The aims of BTE are to regenerate and restore the function of lost bone tissue using combinations of osteoblast progenitors and synthetic biomaterial scaffolds. Over the past decade, the use of synthetic biomaterials to enhance bone regeneration has significantly developed because of their capacity to mimic the natural environment of the extracellular matrix. The synthetic scaffold biomaterials predominantly used in BTE include calcium phosphate ceramics, biodegradable polymers, and composites, and the combination of ceramics and polymer scaffolds aims to utilize the properties of both materials [47,50,51][31][34][35].

3.2.2. Preclinical Studies of BTE in a Large Animal Model Using MSC/Scaffold Combinations

To translate the clinical use of MSCs combined with scaffolds for BTE, large animal model systems that closely resemble human physiology are required. A number of preclinical studies conducted using MSCs with varying combinations of biomaterials in critical bone defect models are listed in Table 1.
Table 1. Pre-clinical experiments of MSCs-combined with biomaterial for bone regeneration in large animal bone defect models.
AuthorExperiment AnimalType and Size of DefectExperimental Transplant GroupsPost-Transplant

Follow up Period		Outcome
Table 3. List of clinical studies listed in clinicaltraisl.gov using MSCs combined with biomaterials for bone tissue regeneration.
NCT NumberBrief TitlePhaseConditionsInterventions
Probst et al., 2020 [34]Mini pigsCritical mandibular defect (3 × 1 × 2 cm)3D TCP-PLGA scaffold seeded with osteogenic differentiated Porcine ADSCs (pADSCs).12 weekspADSCs seeded TCP-PLGA scaffold constructs significantly improved bone regenerations compared to empty scaffold.
NCT04297813Efficacy in Alveolar Bone Regeneration With Autologous MSCs and Biomaterial in Comparison to Autologous Bone GraftingPhase I• Alveolar Bone AtrophyAutologous MSCs and a biomaterial, biphasic Calcium Phosphate (BCP).Wang et al., 2019 [36]Rhesus MonkeysCritical alveolar bone defect (10 × 10 × 5 mm)3D-Bioactive glass (BG) + BMP/chitosan (CS) + BMMSCs12 weeksBMP/CS nanoparticles loaded on 3D-BG scaffold promoted bone regeneration ability in vivo, and preload of BMMSCs promote this ability further.
Humerus, Tibia and Femur Critical sized defectsCombination of HA granules (Bongros®-HA, Bioalpha, Seungnam, Korea), BMP2 and BMMSCs mixed with Plasma solution.Autologous Bone marrow harvested from posterior Iliac crestal boneNoneDramatic improvement of bone regeneration compared to preoperative radiographs.
NCT03325504A Comparative Study of 2 Doses of BM Autologous H- MSC+Biomaterial vs. Iliac Crest AutoGraft for Bone Healing in Non-UnionPhase III• Non Union FractureCulture-expanded autologous BMMSC combined with biphasic calcium phosphate (BCP) biomaterial granulesHsieh et al., 2019 [37]Gjerde et al., 2018 [54domestic Ds-Red pigsCalvarias defect (8 mm in diameter and 2 mm in depth)Hemostatic gelatin sponge scaffold seeded with EGFP pig BMMSCs]Severe mandibular ridge resorption.Expanded, autologous MSCs with biphasic calcium phosphate (MBCP+
NCT028031771, 2, 3 and 4 weeksOsteoid formation in the scaffolds transplanted with seeded BMMSCs was significantly higher than the control group.
Cell Therapy by Autologous BMC for Large Bone Defect RepairTM; Biomatlante, France)Bone marrow cells from the posterior iliac crestShi et al., 2019 [38]MinipigsMaxillary Intraosseous circular defects (12 mm in diameter and 5 mm in depth)Bio-Oss/autogenous (Pig Gingival MSCs) pGMSCs (2 × 106)/SB431542 (TGF-β signalling inhibitor).8 weekspGMSCs treated with a TGF- β signaling inhibitor successfully repair minipig severe maxillofacial bone defects.
NoneMSCs successfully induce significant new bone formationQiu et al., 2018 [39]MinipigsLateral femoral condyle defect (8 mm in diameter and 10 mm in depth)Calcium phosphate cement (CPC) scaffold seeded with autologous BMMSCs plus autologous PRP (CPC-BMSC-PRP, 1 × 106 cells/scaffold)6 and 12 weeksCPC scaffold co-delivered BMMSCs-PRP promoted scaffold resorption and doubled bone regeneration in large defects than control groups
Zhang et al., 2017 [40]MinipigsNon-healing full thickness cranial defects (2 cm width × 3 cm length × 0.5 cm depth)IMC (intrafibrillarly-mineralized collagen) scaffold seeded with 1 × 106 PDLSCs cells12 weeksCompared with HA, IMC-seeded PDLSCs achieved a significantly higher extent of new bone formation, with the normal architecture of natural bones and blood vessels.
Scarano et al., 2017 [41]MinipigsCritical-size circular defects (5 mm diameter; 5 mm thickness) in the mandibular bodyBone porcine block (BPB) scaffold seeded with 100 ul cell suspension of BMMSCs12 weeksBPB when used as a scaffold induce bone regeneration and further benefit from the addition of BMMSCs in the tissue-engineered constructs.
Lin et al., 2015 [42]MinipigsMassive segmental bone defects (30 mm in length) at the mid-diaphysis of femoraTransduced pig ADSCs loaded onto PLGA scaffold2, 4, 8 and 12 weeksADSCs/scaffold constructs successfully healed massive segmental bone defects at the mid-diaphysis of femora in minipigs significantly than control group.
Cao et al., 2015 [43]Mini pigsCalvarial bone defects (3 cm × 1.8 cm oval defect)BMMSCs pretreated with 75 μg/mL aspirin for 24 h seeded onto hydroxyapatite/tricalcium phosphate (HA/TCP)6 monthsBMMSCs pretreated with aspirin have a greater capacity to repair calvarial bone defects in a mini swine model
Fan et al., 2014 [44]Rhesus monkeysSegmental tibial defects (20 mm in length)Autologous prevascularized BMMSCs (5 × 106)-β-TCP constructs4, 8 and

12 weeks		Significantly higher amount of neo-vascularization and radiographic grading score in prevascularized BMMSCs-β-TCP constructs

3.2.3. Gene Therapy for Bone Regeneration

Gene therapy is another promising approach for enhancing bone regeneration. Today, the advancement in life-sciences technology allows gene transfer technology to fabricate a tissue-engineered scaffold to accommodate the growth of genetically modified cells and the endogenous synthesis of desired gene products in a controlled manner. Gene therapy allows for the transfer of genetic material in the precise anatomic location of target cells, allowing the transgene expression from the cells with the currently available techniques [76][45]. Gene transfer can be performed by several ex vivo and in vivo delivery techniques and by either using viral (transduction) or non-viral (transfection) vectors [76,77][45][46]. Since this review is focused on the use of combined cell scaffold constructs for bone regeneration, we mainly discuss the ex vivo delivery method, which requires isolation of target cells and transfer of the desired gene to express the respective protein in vitro and then seeded onto the biocompatible carrier material to obtain cell-scaffold construct for bone tissue engineering applications. The two standard methods of ex vivo delivery include viral and non-viral, it being said that each type has its advantages and disadvantages. Viral vectors demonstrate high transfection efficiency with immunogenicity and toxicity, raising an issue of safety. In contrast, non-viral vectors usually consist of plasmid or related DNA, which are non-immunogenic and high safety but with low transfection efficiency [77,78][46][47]. Another promising approach is the sequential delivery of exogenous genes to promote the osteogenesis of stem cells. For example, genes that are expressed early and in the final stages of osteogenesis are different. Hence, delivering required osteogenic genes at specific time intervals into target cells induces efficient osteogenic differentiation. A recent study by Kim et al. demonstrated an effective sequential delivery of runt-related transcription factor 2 (RUNX2) and osterix genes induced conversion of human MSCs into pre-osteoblasts and subsequent delivery of activating transcription factor 4 (ATF4) gene triggered further osteogenesis. Differentiation of MSCs into desired mature cells can be regulated by the delivery time of specific osteogenic genes mimicking the natural process of bone remodeling [78,79][47][48].

3.2.4. Clinical Trials of MSCs for BTE

Over the past decade, a greater understanding has emerged with regard to the capabilities of MSCs to promote bone tissue regeneration, with numerous preclinical and clinical studies now underway. To identify the current potential combination of cell-scaffold constructs or tissue-engineered substitutes for bone tissue regeneration, we found twenty clinical trials. Nine are published (Table 2), and others are listed in the ClinicalTrails.gov database (Table 3). These trials have highlighted the importance of using cell-based therapy with various scaffolds to treat bone tissue regeneration in a real clinical setting. From the twenty identified clinical studies listed in Table 2 and Table 3, the majority report the use of BMMSCs, reflecting the fact that they are the most accepted cell source and the current gold standard in most clinical trials for treating bone disease, including nonunion fractures of long bones and craniofacial bone defects. However, in a few clinical trials, researchers have used umbilical cord (UC)- MSCs [85][49], BMMSCs [86][50], and adipose-derived MSCs as allogeneic cell sources to prepare the tissue-engineered constructs for regeneration of critical bone defects (NCT02307). Ceramic-based scaffolds are the primary choice in the majority of clinical trials, indicating their high clinical relevance. From the clinical trials listed in Table 2 and Table 3, most studies used a combination of BMMSCs with calcium-phosphate ceramics such as hydroxyapatite [85[49][51][52],87,88], β-TCP [86,89][50][53] (NCT02803177, NCT02153372), biphasic calcium phosphate, a combination of hydroxyapatite and β-TCP [90][54] (NCT04297813, NCT03325504, NCT01842477). Although most of these clinical trials used a simple combination of calcium-phosphate ceramics with BMMSCs, in a few studies, however, additional factors were included to facilitate enhanced bone regeneration. For example, Dilogo et al. added growth factor BMP2 along with cell scaffold constructs to enhance bone regeneration [85,88][49][52]. Similarly, researchers used BMMSCs mixed with BMP2 and loaded them on to 3-dimensional tissue-engineered collagen scaffold (NCT01958502) in another clinical trial. However, a clinical study by Baba et al. used polylactic scaffold seeded BMMSCs mixed with platelet-rich plasma solution and an additional 5000 units of human thrombin dissolved in 10% calcium chloride [91][55].
Table 2. Completed and published clinical studies using MSCs combined with biomaterials for bone tissue regeneration.
AuthorType and Size of DefectTransplant GroupsOrigin of Cell SourcePre-Transplant IncubationOutcome
Dilogo et al.,

2020 [49]		Nonunion fractures of Humerus/tibia with critical size bone defectsCombination of HA Bongros®-HA, Daewoong), BMP2, UC-MSCs with demineralized bone matrixAllogeneic Umbilical Cord MSCs (UC-MSCs)NoneAllogeneic UC-MSCs can be used safely to treat the critical sized bone defects of long bones.
Dilogo et al., 2019 [52]
Phase II• Humerus Fracture Displaced ProximalBaba et al., 2016 [55]Intrabony Periodontal defect. Probing depth >4 mmThe mixture of BMMSCs and PRP, combined with human thrombin dissolved in 10% calcium chloride perfused in a 3D woven-fabric composed of poly-L-lactic acid resin fibers (MSCs/PRP-3D woven Fabric)Autologous Bone marrow harvested from posterior Iliac crestal boneInduced under Osteogenic MediumBMMSCs/PRP-3D woven Fabric constructs showed efficient regeneration of the periodontal tissue including alveolar bone.
Morrison et al., 2018 [50]Cranial defects with less than 80 mm diameterAllogeneic mesenchymal stromal cells (MSCs) on a ceramic carrier (ChronOS granules, synthes, and polymer

scaffold,		Allogenic BMMSCs from 18–25 years aged donorsNoneAllogeneic MSCs can be safely used for bone regeneration.
Autologous Bone Marrow-derived Mononuclear Cells (BMC) seeded onto ß-TCPKaigler et al., 2015 [53]Severe Bone Atrophy of upper JawCombination of BMMSCs and β-TCP (Cerasorb, Curasan AG, Germany)Autologous Bone marrow harvested from posterior Iliac crestal boneNoneHigher density of regenerated bone with MSCs+ β-TCP group was observed than control group.
NCT02307435Marcacci et al., 2007 [51]Humerus, Tibia and ulnar Critical sized defectsCombination of invitro expanded BMMSCs seeded with porous hydroxy apatite scaffolds (Finblock, FinCeramica Srl, Faenza, Italy)Autologous Bone marrow harvested from posterior Iliac crestal boneNoneSignificant healing of the CSDs. Attained long term durability of bone regeneration.
Allogenic Mesenchymal Stem Cell for Bone Defect or Non Union FractureEarly Phase I• Non Union Fracture, Metaphyseal Fibrous DefectAllogeneic MSCs from umbilical cord/bone marrow/adipose combined and HA-CaSo4
NCT02153372Cell Therapy by Bone Marrow- derived Mononuclear Cells (BMC) for Large Bone Defect Repair: Phase-I Clinical TrialPhase I• Humerus Fracture Displaced ProximalAutologous Bone Marrow-derived Mononuclear Cells (BMC) seeded onto ß-TCP
NCT01958502Evaluation the Treatment of Nonunion of Long Bone Fracture of Lower Extremities (Femur and Tibia) Using Mononuclear Stem Cells from the Iliac Wing Within a 3-D Tissue Engineered ScaffoldPhase II• Nonunion of FractureBMMSCs with BMP2 within a 3-D tissue engineered collagen scaffold
NCT01842477Evaluation of Efficacy and Safety of Autologous MSCs Combined to Biomaterials to Enhance Bone HealingPhase I/II• Delayed Union After Fracture of Humerus, Tibial or FemurBMMScs mixed with biphasic calciulm granulesBajada et al., 2007 [56]Tibial non-unionCombination of invitro expanded BMMSCs seeded with calcium sulphate pellets (Stimulan, Biocomposites Ltd., Keele, United Kingdom)Autologous Bone marrow harvested from posterior Iliac crestal boneNoneClinical and radiological healing of nonunion was observed
NCT00250302Autologous Implantation of Mesenchymal Stem Cells for the Treatment of Distal Tibial FracturesPhase I/II• Tibial FractureBMMSCs loaded onto a carrier and implanted locally at the defect siteMorishita et al., 2006 [57]Tibial/femur massive defectsAttachment of invitro expanded BMMSCs-HA granulesAutologous Bone marrow harvested from posterior Iliac crestal boneInduced under Osteogenic Medium
NCT00557635Good integration of BMMSCs-HA constructs to the host bone and increased radiographic density of the defect area.
Osseous Setting Improvement With Co-implantation of Osseous Matrix and Mesenchymal Progenitors Cells From Autologous Bone Marrow
Phase II
• Tibia or Femur Pseudo-arthrosis
Injection of an osseous matrix (osteopure) combined with MSC progenitors from autologous bone marrow.
NCT02177565Autologous Stem Cell Therapy for Fracture Non-union HealingNot available• Non-union of FracturesAutologous BMSCs combined with carrier material
NCT01435434Mononucleotide Autologous Stem Cells and Demineralized Bone Matrix in the Treatment of Non Union/Delayed FracturesNot available• Non Union/Delayed FracturesInjection of Autologous Stem Cells and Demineralized Bone Matrix

4. Osteoblast-Based Bone Tissue Regeneration

In addition to the efforts to increase the bone-forming ability of MSCs as a cell source for bone tissue engineering, the use of osteoblasts that are capable of proliferating before maturing, and that can synthesize and deposit bone extracellular matrix components such as osteocalcin (OCN) and bone sialoprotein (BSP), provides a potential alternative BTE cell source for the treatment of large bone defects. However, since BTE is generally approached using a combination of osteoblasts induced from MSCs on biodegradable scaffolds, the resulting bone forming efficacy will be dependent on the differentiation potential of MSCs into osteoblasts. This could hamper the progress of BTE for treating large bone defects. There are two major mechanisms underlying skeletal development, intramembranous and endochondral ossification. In intramembranous ossification, osteoblast lineage cells, i.e., immature osteoblasts, are formed directly from condensed mesenchymal tissue. Endochondral ossification, by contrast, involves the production of osteochondral progenitors from MSCs that give rise to hyperchondrocytes which activate perichondrial cells to differentiate into immature osteoblasts. From the perspective of BTE, the formation of immature osteoblasts is the convergence point for both types of ossification.

5. Conclusions

Critical bone defects that cannot self-heal without a surgical intervention pose a significant challenge in the field of BTE. Compared to the traditional gold standard approach of using autogenous bone, regenerative methods will typically use either exogenous MSCs or immature osteoblasts seeded onto a bioactive scaffold placed at the defect area to regenerate functional bone. Adult MSCs from bone marrow and adipose tissue have often been used in various clinical studies for bone regeneration. The available data from both preclinical studies and clinical trials have shown promising results when BMMSCs are used as a cell source for bone tissue regeneration. Many clinical studies have also shown the beneficial effects of MSCs and scaffold combinations in bone healing. Although encouraging clinical results have been obtained by transplanting MSCs-scaffold constructs, the exact dosage and route of application remain to be optimized, and the fate of transplanted cells and their mechanisms of action need to be better monitored in more extensive future clinical trials. The development of an alternative immature osteoblast source combined with more effective scaffolds is also anticipated in the future. Immature osteoblasts have the ability to become a potential alternative cell source to adult MSCs, as they are osteogenic lineage-committed cells that enhance the efficacy of bone regeneration.
Immature osteoblasts can be obtained from bone tissue samples during routine oral surgical procedures from mandible/maxillary alveolar bone or surgeries involving long bones from the femur or tibia. The advantage of immature osteoblasts over MSCs is their spontaneous matrix formation upon transplantation without promoting osteogenic differentiation. Immature osteoblasts can directly secrete bone collagen matrix and release various factors such as M-CSF, RANKL, and VEGF, enhancing bone remodeling and bone regeneration in various BTE applications. The combination of an immature osteoblast culture system that possesses robust osteogenic activity and an appropriate biodegradable scaffold is an expected future BTE therapeutic option. This approach will facilitate the establishment of better clinical protocols for regeneration therapy in cases of large bone defects, as a treatment for orthopedic conditions such as back pain resulting from stenosis and lumbar spondylolisthesis osteosarcoma, and for horizontal alveolar bone defects in the dental field.
 
 
 

References

  1. Nauth, A.; Schemitsch, E.; Norris, B.; Nollin, Z.; Watson, J.T. Critical-Size Bone Defects: Is There a Consensus for Diagnosis and Treatment? J. Orthop. Trauma 2018, 32, S7–S11.
  2. Roddy, E.; DeBaun, M.R.; Daoud-Gray, A.; Yang, Y.P.; Gardner, M.J. Treatment of critical-sized bone defects: Clinical and tissue engineering perspectives. Eur. J. Orthop. Surg. Traumatol. 2018, 28, 351–362.
  3. Yuan, H.; Xing, K.; Hsu, H.-Y. Trinity of Three-Dimensional (3D) Scaffold, Vibration, and 3D Printing on Cell Culture Application: A Systematic Review and Indicating Future Direction. Bioengineering 2018, 5, 57.
  4. Plotkin, L.I.; Bellido, T. Osteocytic signalling pathways as therapeutic targets for bone fragility. Nat. Rev. Endocrinol. 2016, 12, 593–605.
  5. Wang, T.; Yang, X.; Qi, X.; Jiang, C. Osteoinduction and proliferation of bone-marrow stromal cells in three-dimensional poly (ε-caprolactone)/ hydroxyapatite/collagen scaffolds. J. Transl. Med. 2015, 13, 152.
  6. Bhumiratana, S.; Bernhard, J.C.; Alfi, D.M.; Yeager, K.; Eton, R.E.; Bova, J.; Shah, F.; Gimble, J.M.; Lopez, M.J.; Eisig, S.B.; et al. Tissue-engineered autologous grafts for facial bone reconstruction. Sci. Transl. Med. 2016, 8, 343ra83.
  7. Chamieh, F.; Collignon, A.-M.; Coyac, B.R.; Lesieur, J.; Ribes, S.; Sadoine, J.; Llorens, A.; Nicoletti, A.; Letourneur, D.; Colombier, M.-L.; et al. Accelerated craniofacial bone regeneration through dense collagen gel scaffolds seeded with dental pulp stem cells. Sci. Rep. 2016, 6, 38814.
  8. Haj, J.; Haj Khalil, T.; Falah, M.; Zussman, E.; Srouji, S. An ECM-Mimicking, Mesenchymal Stem Cell-Embedded Hybrid Scaffold for Bone Regeneration. BioMed Res. Int. 2017, 2017, 1–12.
  9. Kjærgaard, K.; Dreyer, C.H.; Ditzel, N.; Andreasen, C.M.; Chen, L.; Sheikh, S.P.; Overgaard, S.; Ding, M. Bone Formation by Sheep Stem Cells in an Ectopic Mouse Model: Comparison of Adipose, and Bone Marrow Derived Cells and Identification of Donor-Derived Bone by Antibody Staining. Stem Cells Int. 2016, 2016, 1–10.
  10. Nie, L.; Yang, X.; Duan, L.; Huang, E.; Pengfei, Z.; Luo, W.; Zhang, Y.; Zeng, X.; Qiu, Y.; Cai, T.; et al. The healing of alveolar bone defects with novel bio-implants composed of Ad-BMP9-transfected rDFCs and CHA scaffolds. Sci. Rep. 2017, 7, 6373.
  11. Lin, Y.; Umebayashi, M.; Abdallah, M.-N.; Dong, G.; Roskies, M.G.; Zhao, Y.F.; Murshed, M.; Zhang, Z.; Tran, S.D. Combination of polyetherketoneketone scaffold and human mesenchymal stem cells from temporomandibular joint synovial fluid enhances bone regeneration. Sci. Rep. 2019, 9, 472.
  12. Medhat, D.; Rodríguez, C.I.; Infante, A. Immunomodulatory Effects of MSCs in Bone Healing. Int. J. Mol. Sci. 2019, 20, 5467.
  13. Jayakumar, P.; Di Silvio, L. Osteoblasts in bone tissue engineering. Proc. Inst. Mech. Eng. 2010, 224, 1415–1440.
  14. Aino, M.; Nishida, E.; Fujieda, Y.; Orimoto, A.; Mitani, A.; Noguchi, T.; Makino, H.; Murakami, S.; Umezawa, A.; Yoneda, T.; et al. Isolation and characterization of the human immature osteoblast culture system from the alveolar bones of aged donors for bone regeneration therapy. Expert Opin. Biol. Ther. 2014, 14, 1731–1744.
  15. Marolt Presen, D.; Traweger, A.; Gimona, M.; Redl, H. Mesenchymal Stromal Cell-Based Bone Regeneration Therapies: From Cell Transplantation and Tissue Engineering to Therapeutic Secretomes and Extracellular Vesicles. Front. Bioeng. Biotechnol. 2019, 7, 352.
  16. Kanke, K.; Masaki, H.; Saito, T.; Komiyama, Y.; Hojo, H.; Nakauchi, H.; Lichtler, A.C.; Takato, T.; Chung, U.; Ohba, S. Stepwise Differentiation of Pluripotent Stem Cells into Osteoblasts Using Four Small Molecules under Serum-free and Feeder-free Conditions. Stem Cell Rep. 2014, 2, 751–760.
  17. Marolt, D.; Campos, I.M.; Bhumiratana, S.; Koren, A.; Petridis, P.; Zhang, G.; Spitalnik, P.F.; Grayson, W.L.; Vunjak-Novakovic, G. Engineering bone tissue from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 2012, 109, 8705–8709.
  18. Zhu, H.; Kimura, T.; Swami, S.; Wu, J.Y. Pluripotent stem cells as a source of osteoblasts for bone tissue regeneration. Biomaterials 2019, 196, 31–45.
  19. Morikawa, S.; Mabuchi, Y.; Niibe, K.; Suzuki, S.; Nagoshi, N.; Sunabori, T.; Shimmura, S.; Nagai, Y.; Nakagawa, T.; Okano, H.; et al. Development of mesenchymal stem cells partially originate from the neural crest. Biochem. Biophys. Res. Commun. 2009, 379, 1114–1119.
  20. Caplan, A.I. Adult mesenchymal stem cells and the NO pathways. Proc. Natl. Acad. Sci. USA 2013, 110, 2695–2696.
  21. Caplan, A.I. Adult Mesenchymal Stem Cells: When, Where, and How. Stem Cells Int. 2015, 2015, 1–6.
  22. Sousa, B.R.; Parreira, R.C.; Fonseca, E.A.; Amaya, M.J.; Tonelli, F.M.; Lacerda, S.M.; Lalwani, P.; Santos, A.K.; Gomes, K.N.; Ulrich, H.; et al. Human adult stem cells from diverse origins: An overview from multiparametric immunophenotyping to clinical applications. Cytometry A 2014, 85, 43–77.
  23. Mushahary, D.; Spittler, A.; Kasper, C.; Weber, V.; Charwat, V. Isolation, cultivation, and characterization of human mesenchymal stem cells. Cytometry A 2018, 93, 19–31.
  24. Nugraha, A.P.; Rantam, F.A.; Narmada, I.B.; Ernawati, D.S.; Ihsan, I.S. Gingival-Derived Mesenchymal Stem Cell from Rabbit (Oryctolagus cuniculus): Isolation, Culture, and Characterization. Eur. J. Dent. 2021, 15, 332–339.
  25. Chan, B.P.; Leong, K.W. Scaffolding in tissue engineering: General approaches and tissue-specific considerations. Eur. Spine J. 2008, 17, 467–479.
  26. Yu, X.; Tang, X.; Gohil, S.V.; Laurencin, C.T. Biomaterials for bone regenerative engineering. Adv. Healthc. Mater. 2015, 4, 1268–1285.
  27. Kim, Y.; Ko, H.; Kwon, I.K.; Shin, K. Extracellular matrix revisited: Roles in tissue engineering. Int. Neurourol. J. 2016, 20, S23.
  28. Vasanthan, J.; Gurusamy, N.; Rajasingh, S.; Sigamani, V.; Kirankumar, S.; Thomas, E.L.; Rajasingh, J. Role of Human Mesenchymal Stem Cells in Regenerative Therapy. Cells 2020, 10, 54.
  29. Chocholata, P.; Kulda, V.; Babuska, V. Fabrication of Scaffolds for Bone-Tissue Regeneration. Materials 2019, 12, 568.
  30. Zhu, L.; Luo, D.; Liu, Y. Effect of the nano/microscale structure of biomaterial scaffolds on bone regeneration. Int. J. Oral Sci. 2020, 12, 6.
  31. Alizadeh-Osgouei, M.; Li, Y.; Wen, C. A comprehensive review of biodegradable synthetic polymer-ceramic composites and their manufacture for biomedical applications. Bioact. Mater. 2019, 4, 22–36.
  32. Gao, C.; Peng, S.; Feng, P.; Shuai, C. Bone biomaterials and interactions with stem cells. Bone Res. 2017, 5, 1–33.
  33. Girón, J.; Kerstner, E.; Medeiros, T.; Oliveira, L.; Machado, G.; Malfatti, C.; Pranke, P. Biomaterials for bone regeneration: An orthopedic and dentistry overview. Braz. J. Med. Biol. Res. 2021, 54, e11055.
  34. Probst, F.A.; Fliefel, R.; Burian, E.; Probst, M.; Eddicks, M.; Cornelsen, M.; Riedl, C.; Seitz, H.; Aszódi, A.; Schieker, M.; et al. Bone regeneration of minipig mandibular defect by adipose derived mesenchymal stem cells seeded tri-calcium phosphate- poly(D,L-lactide-co-glycolide) scaffolds. Sci. Rep. 2020, 10, 2062.
  35. Amin, A.M.M.; Ewais, E.M.M. Bioceramic Scaffolds. In Scaffolds in Tissue Engineering Materials, Technologies and Clinical Applications; Baino, F., Ed.; InTech: London, UK, 2017.
  36. Wang, L.; Xu, W.; Chen, Y.; Wang, J. Alveolar bone repair of rhesus monkeys by using BMP-2 gene and mesenchymal stem cells loaded three-dimensional printed bioglass scaffold. Sci. Rep. 2019, 9, 18175.
  37. Hsieh, M.-K.; Wu, C.-J.; Su, X.-C.; Chen, Y.-C.; Tsai, T.-T.; Niu, C.-C.; Lai, P.-L.; Wu, S.-C. Bone regeneration in Ds-Red pig calvarial defect using allogenic transplantation of EGFP-pMSCs—A comparison of host cells and seeding cells in the scaffold ed G Papaccio. PLoS ONE 2019, 14, e0215499.
  38. Shi, A.; Heinayati, A.; Bao, D.; Liu, H.; Ding, X.; Tong, X.; Wang, L.; Wang, B.; Qin, H. Small molecule inhibitor of TGF-β signaling enables robust osteogenesis of autologous GMSCs to successfully repair minipig severe maxillofacial bone defects. Stem Cell Res. Ther. 2019, 10, 172.
  39. Qiu, G.; Shi, Z.; Xu, H.H.K.; Yang, B.; Weir, M.D.; Li, G.; Song, Y.; Wang, J.; Hu, K.; Wang, P.; et al. Bone regeneration in minipigs via calcium phosphate cement scaffold delivering autologous bone marrow mesenchymal stem cells and platelet-rich plasma. J. Tissue Eng. Regen. Med. 2018, 12, e937–e948.
  40. Zhang, C.; Yan, B.; Cui, Z.; Cui, S.; Zhang, T.; Wang, X.; Liu, D.; Yang, R.; Jiang, N.; Zhou, Y.; et al. Bone regeneration in minipigs by intrafibrillarly-mineralized collagen loaded with autologous periodontal ligament stem cells. Sci. Rep. 2017, 7, 10519.
  41. Scarano, A.; Crincoli, V.; Di Benedetto, A.; Cozzolino, V.; Lorusso, F.; Podaliri Vulpiani, M.; Grano, M.; Kalemaj, Z.; Mori, G.; Grassi, F.R. Bone Regeneration Induced by Bone Porcine Block with Bone Marrow Stromal Stem Cells in a Minipig Model of Mandibular “Critical Size” Defect. Stem Cells Int. 2017, 2017, 9082869.
  42. Lin, C.-Y.; Wang, Y.-H.; Li, K.-C.; Sung, L.-Y.; Yeh, C.-L.; Lin, K.-J.; Yen, T.-C.; Chang, Y.-H.; Hu, Y.-C. Healing of massive segmental femoral bone defects in minipigs by allogenic ASCs engineered with FLPo/Frt-based baculovirus vectors. Biomaterials 2015, 50, 98–106.
  43. Cao, Y.; Xiong, J.; Mei, S.; Wang, F.; Zhao, Z.; Wang, S.; Liu, Y. Aspirin promotes bone marrow mesenchymal stem cell-based calvarial bone regeneration in mini swine. Stem Cell Res. Ther. 2015, 6, 210.
  44. Fan, H.; Zeng, X.; Wang, X.; Zhu, R.; Pei, G. Efficacy of prevascularization for segmental bone defect repair using β-tricalcium phosphate scaffold in rhesus monkey. Biomaterials 2014, 35, 7407–7415.
  45. Dimitriou, R.; Jones, E.; McGonagle, D.; Giannoudis, P.V. Bone regeneration: Current concepts and future directions. BMC Med. 2011, 9, 66.
  46. Evans, C. Gene therapy for the regeneration of bone. Injury 2011, 42, 599–604.
  47. Franceschi, R.T.; Yang, S.; Rutherford, R.B.; Krebsbach, P.H.; Zhao, M.; Wang, D. Gene Therapy Approaches for Bone Regeneration. Cells Tissues Organs 2004, 176, 95–108.
  48. Kim, H.J.; Park, J.S.; Yi, S.W.; Oh, H.J.; Kim, J.-H.; Park, K.-H. Sequential transfection of RUNX2/SP7 and ATF4 coated onto dexamethasone-loaded nanospheres enhances osteogenesis. Sci. Rep. 2018, 8, 1447.
  49. Dilogo, I.H.; Rahmatika, D.; Pawitan, J.A.; Liem, I.K.; Kurniawati, T.; Kispa, T.; Mujadid, F. Allogeneic umbilical cord-derived mesenchymal stem cells for treating critical-sized bone defects: A translational study. Eur. J. Orthop. Surg. Traumatol. 2021, 31, 265–273.
  50. Morrison, D.A.; Kop, A.M.; Nilasaroya, A.; Sturm, M.; Shaw, K.; Honeybul, S. Cranial reconstruction using allogeneic mesenchymal stromal cells: A phase 1 first-in-human trial. J. Tissue Eng. Regen. Med. 2018, 12, 341–348.
  51. Marcacci, M.; Kon, E.; Moukhachev, V.; Lavroukov, A.; Kutepov, S.; Quarto, R.; Mastrogiacomo, M.; Cancedda, R. Stem Cells Associated with Macroporous Bioceramics for Long Bone Repair: 6- to 7-Year Outcome of a Pilot Clinical Study. Tissue Eng. 2007, 13, 947–955.
  52. Dilogo, I.H.; Phedy, P.; Kholinne, E.; Djaja, Y.P.; Fiolin, J.; Kusnadi, Y.; Yulisa, N.D. Autologous mesenchymal stem cell implantation, hydroxyapatite, bone morphogenetic protein-2, and internal fixation for treating critical-sized defects: A translational study. Int. Orthop. 2019, 43, 1509–1519.
  53. Kaigler, D.; Avila-Ortiz, G.; Travan, S.; Taut, A.D.; Padial-Molina, M.; Rudek, I.; Wang, F.; Lanis, A.; Giannobile, W.V. Bone Engineering of Maxillary Sinus Bone Deficiencies Using Enriched CD90+ Stem Cell Therapy: A Randomized Clinical Trial: Stem cell therapy for advanced maxillary bone defects. J. Bone Miner. Res. 2015, 30, 1206–1216.
  54. Gjerde, C.; Mustafa, K.; Hellem, S.; Rojewski, M.; Gjengedal, H.; Yassin, M.A.; Feng, X.; Skaale, S.; Berge, T.; Rosen, A.; et al. Cell therapy induced regeneration of severely atrophied mandibular bone in a clinical trial. Stem Cell Res. Ther. 2018, 9, 213.
  55. Baba, S.; Yamada, Y.; Komuro, A.; Yotsui, Y.; Umeda, M.; Shimuzutani, K.; Nakamura, S. Phase I/II Trial of Autologous Bone Marrow Stem Cell Transplantation with a Three-Dimensional Woven-Fabric Scaffold for Periodontitis. Stem Cells Int. 2016, 2016, 1–7.
  56. Bajada, S.; Harrison, P.E.; Ashton, B.A.; Cassar-Pullicino, V.N.; Ashammakhi, N.; Richardson, J.B. Successful treatment of refractory tibial nonunion using calcium sulphate and bone marrow stromal cell implantation. J. Bone Jt. Surg. Br. 2007, 89-B, 1382–1386.
  57. Morishita, T.; Honoki, K.; Ohgushi, H.; Kotobuki, N.; Matsushima, A.; Takakura, Y. Tissue Engineering Approach to the Treatment of Bone Tumors: Three Cases of Cultured Bone Grafts Derived From Patients’ Mesenchymal Stem Cells. Artif. Organs 2006, 30, 115–118.
More
© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)
Video Production Service
Feedback